Acuaporinas en diversas patologías neurológicas

El agua es un compuesto esencial para la vida. La mayoría de las moléculas (iones, azúcares, proteínas, etc.) que forman parte de los seres vivos se encuentran disueltos en medio acuoso. Para realizar sus funciones, las células intercambian moléculas con su entorno a través de la membrana, mediante mecanismos de difusión pasiva o mediante proteínas transportadoras específicas, las acuaporinas. La expresión de acuaporinas ha sido estudiada ampliamente en el sistema nervioso central, órganos sensoriales y sistema nervioso entérico y periférico. Su estudio es de gran relevancia debido a la implicación en la etiología de diferentes patologías neurológicas, siendo AQP1, AQP4 y AQP9 las descritas en el SNC. El conocimiento de las funciones de las acuaporinas en el SNC se derivan en gran parte de experimentos realizados en ratones knockout de AQPs (AQP-KO) para diferentes condiciones patológicas, puesto que hasta la fecha no se han descrito inhibidores de AQPs que no sean tóxicos para el organismo y tampoco han sido identificadas en humanos mutaciones en AQPs que conlleven pérdida de función de éstas. Al menos tres grupos han generado ratones AQP4-KO que han permitido describir tres principales funciones como son el facilitar el paso de agua hacia dentro y hacia fuera del SNC, migración de astrocitos y neuroexcitación. Dentro de las patologías que se relacionan con las acuaporinas encontramos: la formación de edemas, tumores cerebrales, epilepsia, neuromielitis óptica (NMO) y la hidrocefalia. Estas dos últimas atañen especialmente al trabajo realizado en esta Tesis Doctoral. La neuromielitis óptica (NMO), también conocida como enfermedad de Devic’s, es una enfermedad autoinmune inflamatoria de carácter desmielinizante del sistema nervioso central que principalmente afecta al nervio óptico y a la médula espinal. Durante mucho tiempo ha existido mucha controversia de si la neuromielitis óptica es una variante de la esclerosis múltiple (EM) o es una enfermedad diferente. Sin embargo gracias a estudios clínicos, inmunológicos, radiológicos y patológicos, se ha establecido que la NMO es una enfermedad distinta de la EM, viéndose aún más reforzada esta postura tras el descubrimiento, en 2004, de un anticuerpo específico anti-AQP4 en pacientes con NMO. La hidrocefalia es una afección en la que un exceso de líquido cefalorraquídeo se acumula en los ventrículos del cerebro. En condiciones normales existe un equilibrio entre la producción, la circulación y los niveles de absorción de líquido cefalorraquídeo. La hidrocefalia es el resultado de un desequilibrio en estos procesos que lleva a un incremento del volumen ocupado por este fluido en el SNC. Existen 2 tipos de hidrocefalias que atendiendo al criterio del flujo del LCR se clasifican en: hidrocefalia comunicante e hidrocefalia obstructiva. Existen pocos estudios que relacionan AQP1 con la hidrocefalia, donde la mayoría se centra en modelos animales de hidrocefalia congénitos o inducidos. En humanos, aunque no queda claro el patrón de expresión de AQP1 en hidrocefalia, la mayoría de estudios apuesta por una diminución poco significativa de la expresión, sugiriendo ser un mecanismo adaptativo que actúa para disminuir la producción de LCR y la presión intracraneal. Dos grandes Objetivos orientaron el trabajo que aquí se presenta. - El primero fue entender el papel de AQP4 en la patología de la Neuromielitis óptica (NMO), y desarrollar un sistema de diagnóstico, específico, sensible, rápido y cuantitativo, para la NMO mediante la detección de anticuerpos anti-AQP4 (y anti AQP1) en suero de pacientes. El método CBA (Cell Based Assay), fue el método que desarrollamos para AQP4 por presentar los mejores resultados de especificidad y sensibilidad. Sin embargo, AQP1 no consideramos que sea un buen marcador de la enfermedad por el resultado negativo obtenido en la detección de anticuerpos en suero de pacientes con NMOSD. - El segundo fue estudiar la participación de las principales acuaporinas de cerebro en la homeostasis del líquido cefalorraquídeo (LCR) en situación control y bajo el estímulo hipóxico; y discernir el posible papel de estas proteínas en el desarrollo de la hidrocefalia, usando modelos animales y analizando muestras de LCR de humanos. Confirmamos el papel clave de las acuaporinas en la homeostasis del LCR en cerebro y la posible implicación de AQP1 y AQP4 en el desarrollo de la hidrocefalia, siendo de especial importancia el tejido ependimario y el plexo coroideo

Nrf2 and Heme Oxygenase-1 Involvement in Atherosclerosis Related Oxidative Stress

Atherosclerosis remains the underlying process responsible for cardiovascular diseases and the high mortality rates associated. This chronic inflammatory disease progresses with the formation of occlusive atherosclerotic plaques over the inner walls of vascular vessels, with oxidative stress being an important element of this pathology. Oxidation of low-density lipoproteins (ox-LDL) induces endothelial dysfunction, foam cell activation, and inflammatory response, resulting in the formation of fatty streaks in the atherosclerotic wall. With this in mind, different approaches aim to reduce oxidative damage as a strategy to tackle the progression of atherosclerosis. Special attention has been paid in recent years to the transcription factor Nrf2 and its downstream-regulated protein heme oxygenase-1 (HO-1), both known to provide protection against atherosclerotic injury. In the current review, we summarize the involvement of oxidative stress in atherosclerosis, focusing on the role that these antioxidant molecules exert, as well as the potential therapeutic strategies applied to enhance their antioxidant and antiatherogenic properties

An immunoassay that distinguishes real neuromyelitis optica signals from a labeling detected in patients receiving natalizumab

BACKGROUND: Cell-based assays for neuromyelitis optica (NMO) diagnosis are the most sensitive and specific methods to detect anti-aquaporin 4 (AQP4) antibodies in serum, but some improvements in their quantitative and specificity capacities would be desirable. Thus the aim of the present work was to develop a sensitive quantitative method for detection of anti-AQP4 antibodies that allows clear diagnosis of NMO and distinction of false labeling produced by natalizumab treatment. METHODS: Sera from 167 individuals, patients diagnosed with NMO (16), multiple sclerosis (85), optic neuritis (24), idiopathic myelitis (21), or other neurological disorders (13) and healthy controls (8), were used as the primary antibody in an immunofluorescence assay on HEK cells transfected with the M23 isoform of human AQP4 fused with enhanced green fluorescent protein. Cells used were freshly transfected or stored frozen and then thawed just before adding the serum. RESULTS: Microscopic observation and fluorescence quantification produced similar results in fresh and frozen samples. Serum samples from patients diagnosed with NMO were 100% positive for anti-AQP4 antibodies, while all the other sera were negative. Using serum from patients treated with natalizumab, a small and unspecific fluorescent signal was produced from all HEK cells, regardless of AQP4 expression. CONCLUSIONS: Our cell-based double-label fluorescence immunoassay protocol significantly increases the signal specificity and reduces false diagnosis of NMO patients, especially in those receiving natalizumab treatment. Frozen pretreated cells allow faster detection of anti-AQP4 antibodies

Comparative Analysis for the Presence of IgG Anti-Aquaporin-1 in Patients with NMO-Spectrum Disorders

Detection of IgG anti-Aquaporin-4 (AQP4) in serum of patients with Neuromyelitis optica syndrome disorders (NMOSD) has improved diagnosis of these processes and differentiation from Multiple sclerosis (MS). Recent findings also claim that a subgroup of patients with NMOSD, serum negative for IgG-anti-AQP4, present antibodies anti-AQP1 instead. Explore the presence of IgG-anti-AQP1 using a previously developed cell-based assay (CBA) highly sensitive to IgG-anti-AQP4. Serum of 205 patients diagnosed as NMOSD (8), multiple sclerosis (94), optic neuritis (39), idiopathic myelitis (29), other idiopathic demyelinating disorders of the central nervous system (9), other neurological diseases (18) and healthy controls (8), were used in a CBA over fixed HEK cells transfected with hAQP1-EGFP or hM23-AQP4-EGFP, treated with Triton X-100 and untreated. ELISA was also performed. Analysis of serum with our CBA indicated absence of anti-AQP1 antibodies, whereas in cells pretreated with detergent, noisy signal made reliable detection impossible. ELISA showed positive results in few serums. The low number of NMOSD serums included in our study reduces its power to conclude the specificity of AQP1 antibodies as new biomarkers of NMOSD. Our study does not sustain detection of anti-AQP1 in serum of NMOSD patients but further experiments are expected.Grants from “La Junta de Andalucía, Consejería de Innovación Ciencia y Empresa” (P08-CTS-03574) and Consejería de Salud (PI0298-2010), and from the “Instituto de Salud Carlos III” (Exp. PI12/01882) to Miriam Echevarría funded this work. We thank Genzyme Foundation in multiple sclerosis for giving to Miriam Echevarría one of their 2012 fellowships. We acknowledge support by the CSIC Open Access Publication Initiative through its Unit of Information Resources for Research (URICI

The Role of Microglia in Glioblastoma

Glioblastoma (GB), the most aggressive malignant glioma, is made up of a large percentage of glioma-associated microglia/macrophages (GAM), suggesting that immune cells play an important role in the pathophysiology of GB. Under physiological conditions, microglia, the phagocytes of the central nervous system (CNS), are involved in various processes such as neurogenesis or axonal growth, and the progression of different conditions such as Alzheimer's disease. Through immunohistochemical studies, markers that enhance GB invasiveness have been shown to be expressed in the peritumoral area of the brain, such as Transforming Growth Factor alpha (TGF-alpha), Stromal Sell-Derived Factor 1 (SDF1/CXCL12), Sphingosine-1-Phosphate (S1P) and Neurotrophic Factor Derived from the Glial cell line (GDNF), contributing to the increase in tumor mass. Similarly, it has also been described 17 biomarkers that are present in hypoxic periarteriolar HSC niches in bone marrow and in hypoxic periarteriolar GSC niches in glioblastoma. Interestingly, microglia plays an important role in the microenvironment that supports GB progression, being one of the most important focal points in the study of therapeutic targets for the development of new drugs. In this review, we describe the altered signaling pathways in microglia in the context of GB. We also show how microglia interact with glioblastoma cells and the epigenetic mechanisms involved. Regarding the interactions between microglia and neurogenic niches, some authors indicate that glioblastoma stem cells (GSC) are similar to neural stem cells (NSC), common stem cells in the subventricular zone (SVZ), suggesting that this could be the origin of GB. Understanding the similarities between SVZ and the tumor microenvironment could be important to clarify some mechanisms involved in GB malignancy and to support the discovering of new therapeutic targets for the development of more effective glioblastoma treatments

New insight in Human Anatomical teaching for Sport Science students

Al impartir por primera vez Anatomía en el grado en Ciencias de la Actividad Física y Deporte, nos enfrentamos al descontento del alumnado, pues consideraban la Anatomía como una asignatura compleja y sin interés para ellos. Por eso, nos planteamos desarrollar un modelo motivacional y diseñamos prácticas específicas donde estudiar un grupo muscular determinado a través de un deporte seleccionado (futbol, baloncesto, golf, tenis o running). Además, implantamos otra serie de dinámicas que aumentaron el interés por la Anatomía, como la gamificación y la utilización de programas de simulación en 3D. Realizamos cuestionarios de conocimientos antes y después de la experiencia y una encuesta de satisfacción final. Tras el análisis de los resultados, observamos la efectividad de nuestra herramienta y demostramos que, gracias a las prácticas específicas, disminuyó el grado de errores en los cuestionarios realizados. Tanto esta modalidad de docencia, como la gamificación, fueron ampliamente aceptadas por el alumnado.When teaching Anatomy for the first time in Physical Activity and Sports Science Degree we faced with the discontent of the students, as they considered Anatomy as a complex subject and without interest for them. For this reason, we proposed to development a motivational model and design specific practices where to study a selected group of muscles through with a specific sport (football, basketball, golf and running). In addition, we implemented another series of teaching dynamics in order to increase the interest in Anatomy, such as gamification and 3D simulation programs. We carry out knowledge questionnaires before and after the experience and also, a final satisfaction survey. After analyzing the results, we observed the effectiveness of our tool and demonstrated that, thanks to the specific practices, the made mistakes were decreased in the questionaries. This teaching modality plus the use of gamification were widely accepted by the student

Comparative analysis for the presence of IgG anti-aquaporin-1 in patients with NMO-Spectrum disorders

Detection of IgG anti-Aquaporin-4 (AQP4) in serum of patients with Neuromyelitis optica syndrome disorders (NMOSD) has improved diagnosis of these processes and differentiation from Multiple sclerosis (MS). Recent findings also claim that a subgroup of patients with NMOSD, serum negative for IgG-anti-AQP4, present antibodies anti-AQP1 instead. Explore the presence of IgG-anti-AQP1 using a previously developed cell-based assay (CBA) highly sensitive to IgG-anti-AQP4. Serum of 205 patients diagnosed as NMOSD (8), multiple sclerosis (94), optic neuritis (39), idiopathic myelitis (29), other idiopathic demyelinating disorders of the central nervous system (9), other neurological diseases (18) and healthy controls (8), were used in a CBA over fixed HEK cells transfected with hAQP1-EGFP or hM23-AQP4-EGFP, treated with Triton X-100 and untreated. ELISA was also performed. Analysis of serum with our CBA indicated absence of anti-AQP1 antibodies, whereas in cells pretreated with detergent, noisy signal made reliable detection impossible. ELISA showed positive results in few serums. The low number of NMOSD serums included in our study reduces its power to conclude the specificity of AQP1 antibodies as new biomarkers of NMOSD. Our study does not sustain detection of anti-AQP1 in serum of NMOSD patients but further experiments are expected

REX-001, a BM-MNC Enriched Solution, Induces Revascularization of Ischemic Tissues in a Murine Model of Chronic Limb-Threatening Ischemia

Background: Bone Marrow Mononuclear Cells (BM-MNC) constitute a promising alternative for the treatment of Chronic Limb-Threatening ischemia (CLTI), a disease characterized by extensive blockade of peripheral arteries, clinically presenting as excruciating pain at rest and ischemic ulcers which may lead to gangrene and amputation. BM-MNC implantation has shown to be efficient in promoting angiogenesis and ameliorating ischemic symptoms in CLTI patients. However, the variability seen between clinical trials makes necessary a further understanding of the mechanisms of action of BM-MNC, and moreover, to improve trial characteristics such as endpoints, inclusion/exclusion criteria or drug product compositions, in order to implement their use as stem-cell therapy. Materials: Herein, the effect of REX-001, a human-BM derived cell suspension enriched for mononuclear cells, granulocytes and CD34+ cells, has been assessed in a murine model of CLTI. In addition, a REX-001 placebo solution containing BM-derived red blood cells (BM-RBCs) was also tested. Thus, 24 h after double ligation of the femoral artery, REX-001 and placebo were administrated intramuscularly to Balb-c nude mice (n:51) and follow-up of ischemic symptoms (blood flow perfusion, motility, ulceration and necrosis) was carried out for 21 days. The number of vessels and vascular diameter sizes were measured within the ischemic tissues to evaluate neovascularization and arteriogenesis. Finally, several cell-tracking assays were performed to evaluate potential biodistribution of these cells. Results: REX-001 induced a significant recovery of blood flow by increasing vascular density within the ischemic limbs, with no cell translocation to other organs. Moreover, cell tracking assays confirmed a decrease in the number of infused cells after 2 weeks post-injection despite on-going revascularization, suggesting a paracrine mechanism of action. Conclusion: Overall, our data supported the role of REX-001 product to improve revascularization and ischemic reperfusion in CLTI

The Role of Glycosyltransferases in Colorectal Cancer

Colorectal cancer (CRC) is one of the main causes of cancer death in the world. Posttranslational modifications (PTMs) have been extensively studied in malignancies due to its relevance in tumor pathogenesis and therapy. This review is focused on the dysregulation of glycosyltransferase expression in CRC and its impact in cell function and in several biological pathways associated with CRC pathogenesis, prognosis and therapeutic approaches. Glycan structures act as interface molecules between cells and their environment and in several cases facilitate molecule function. CRC tissue shows alterations in glycan structures decorating molecules, such as annexin-1, mucins, heat shock protein 90 (Hsp90), 1 integrin, carcinoembryonic antigen (CEA), epidermal growth factor receptor (EGFR), insulin-like growth factor-binding protein 3 (IGFBP3), transforming growth factor beta (TGF- ) receptors, Fas (CD95), PD-L1, decorin, sorbin and SH3 domain-containing protein 1 (SORBS1), CD147 and glycosphingolipids. All of these are described as key molecules in oncogenesis and metastasis. Therefore, glycosylation in CRC can affect cell migration, cell–cell adhesion, actin polymerization, mitosis, cell membrane repair, apoptosis, cell differentiation, stemness regulation, intestinal mucosal barrier integrity, immune system regulation, T cell polarization and gut microbiota composition; all such functions are associated with the prognosis and evolution of the disease. According to these findings, multiple strategies have been evaluated to alter oligosaccharide processing and to modify glycoconjugate structures in order to control CRC progression and prevent metastasis. Additionally, immunotherapy approaches have contemplated the use of neo-antigens, generated by altered glycosylation, as targets for tumor-specific T cells or engineered CAR (Chimeric antigen receptors) T cells

Atherosclerotic Pre-Conditioning Affects the Paracrine Role of Circulating Angiogenic Cells Ex-Vivo

In atherosclerosis, circulating angiogenic cells (CAC), also known as early endothelial progenitor cells (eEPC), are thought to participate mainly in a paracrine fashion by promoting the recruitment of other cell populations such as late EPC, or endothelial colony-forming cells (ECFC), to the injured areas. There, ECFC replace the damaged endothelium, promoting neovascularization. However, despite their regenerative role, the number and function of EPC are severely affected under pathological conditions, being essential to further understand how these cells react to such environments in order to implement their use in regenerative cell therapies. Herein, we evaluated the effect of direct incubation ex vivo of healthy CAC with the secretome of atherosclerotic arteries. By using a quantitative proteomics approach, 194 altered proteins were identified in the secretome of pre-conditioned CAC, many of them related to inhibition of angiogenesis (e.g., endostatin, thrombospondin-1, fibulins) and cell migration. Functional assays corroborated that healthy CAC released factors enhanced ECFC angiogenesis, but, after atherosclerotic pre-conditioning, the secretome of pre-stimulated CAC negatively affected ECFC migration, as well as their ability to form tubules on a basement membrane matrix assay. Overall, we have shown here, for the first time, the effect of atherosclerotic factors over the paracrine role of CAC ex vivo. The increased release of angiogenic inhibitors by CAC in response to atherosclerotic factors induced an angiogenic switch, by blocking ECFC ability to form tubules in response to pre-conditioned CAC. Thus, we confirmed here that the angiogenic role of CAC is highly affected by the atherosclerotic environment